Little is known about the mechanisms by which the cells of mineralized tissues regulate intracellular and extracellular calcium balance. This application is designed to test the hypothesis that osteoblasts cycle Ca2+ across the plasma membrane (PM) in response to the calcitropic steroid, 1alpha,25- dihydroxyvitamin D3 (1,25(OH)2D3). Ca2+ cycling may allow a sustained response to hormones while protecting cells from Ca2+ toxicity. In this model, agents such as 1,25(OH)2D3 activate Ca2+ influx through PM channels while other PM transporters extrude Ca2+. A corollary to this postulate is that Ca2+ cycling prevents long term increases in bulk intracellular free Ca2+ ([Ca2+]i), but leads to localized elevations in (Ca2+]i beneath the PM. The experiments in this application involve MC3T3-E1 cells as an osteoblast model, and the proposal consists of three specific aims. Specific Aim 1 will test our hypothesis that PM Ca2+-ATPase is the main component of Ca2+ efflux in osteoblastic cells. [Ca2+]i will be measured in cells treated with 1,25(OH)2D3 and inhibitors of PM Ca2+-ATPase activity. Previous work has shown that 1,25(OH)2D3 activates L-type channels in osteoblast-like cells without increasing bulk [Ca2+]i. The Ca2+ cycling model predicts that an elevation in bulk [Ca2+]i will be evident in 1,25(OH)2D3 treated cells that have diminished PM Ca2+-ATPase activity. Specific Aim 2 is a test of our hypothesis that Na+/Ca2+-exchange is not a major contributor to the Ca2+ efflux component of Ca2+ cycling. Analogous to the experiments in Specific Aim 1, Na+/Ca2+-exchange in 1,25(OH)2D3-treated cells will be blocked. The prediction is that, as in control cells, bulk [Ca2+]i will not change in response to 1,25(OH)2D3. Specific Aim 3 will employ FFP-18, a membrane-associated form of the Ca2+-sensitive fluorochrome, fura 2, to determine if 1,25(OH)2D3 evokes focal (near the PM) elevations in [Ca2+]i. Together, these experiments will reveal whether MC3T3-E1 osteoblastic cells employ Ca2+ cycling across the PM as a response to physiological agents that regulate bone calcium metabolism.